Suppression of insulin target gene expression by SecinH3 quantified by real-time PCR

doi:10.1038/nprot.2006.413

This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.

Method Article

+1

Suppression of insulin target gene expression by SecinH3 quantified by real-time PCR

Markus Hafner, Anton Schmitz, Michael Famulok

Markus Hafner

LIMES Program Unit Chemical Biology & Medicinal Chemistry, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn

Anton Schmitz

LIMES Program Unit Chemical Biology & Medicinal Chemistry, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn

Michael Famulok

LIMES Program Unit Chemical Biology & Medicinal Chemistry, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn

DOI:

10.1038/nprot.2006.413

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

SecinH3 causes insulin resistance in mouse liver and in cell culture by inhibiting cytohesins that are effectors of the insulin signalling pathway. Stimulation with insulin leads to increased transcription of glycolytic genes, to suppressed transcription of gluconeogenetic genes and IGFBP1 in liver and in HepG2 cells. These insulin effects can be reverted by SecinH3. We quantified gene expression levels by real-time PCR.

10 mg mouse liver (or ca. 10⁶-10⁷ HepG2 cells grown to 75% confluence)

SecinH3 (100 mM in DMSO)

D5 (10 mM in DMSO)

DMSO

insulin (Sigma)

standard cell culture material and media

RNA-extraction kit (Absolutely RNA, Stratagene)

RT-kit (cDNA-Archive kit, Applied Biosystems)

TaqMan probes for assayed genes (Applied Biosystems)

TaqMan MasterMix, 2x (Applied Biosystems)

Standard cell culture equipment

Real-time PCR machine (BioRad)

A: HepG2 cells

ca. 10⁵ HepG2 cells are seeded into wells of a 12-well culture plate and incubated overnight
after washing twice with PBS, the cells are serum starved for 24 hrs
compounds are added in desired concentrations (SecinH3, D5 and Wortmannin or DMSO alone); final concentration of DMSO was 0.2% in our experiment
cells stimulated with 10 nM insulin for 12 hrs
Total RNA extracted with Absolutely RNA kit (Stratagene) according to instructions from manufacturer; elution volume 50 µl

B: Mouse liver

10 mg liver homogenized in lysis buffer and RNA extracted with Absolutely RNA kit (Stratagene) according to instructions of manufacturer; elution volume 50 µl

RT and real-time PCR

RNA concentration determined by photometry
2.5 µg RNA used for reverse transcription with cDNA Archive kit (Applied Biosystems) according to instructions from manufacturer; RT volume: 50 µl
resulting cDNA diluted 1:3 with H₂O
quantitative PCR performed in 10 µl scale with gene-specific TaqMan probes (Applied Biosystems). All expression-levels were normalized to the expression level of β-2-microglobulin.

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Subject Areas

Genetics

Molecular Biology

Biological techniques

Associated Publications

Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance
by Hafner, M. et al.
Nature (16 November, 2006)

Method Article

Genetics

Molecular Biology

+1

Suppression of insulin target gene expression by SecinH3 quantified by real-time PCR

Markus Hafner, Anton Schmitz, Michael Famulok

Markus Hafner

LIMES Program Unit Chemical Biology & Medicinal Chemistry, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn

Anton Schmitz

LIMES Program Unit Chemical Biology & Medicinal Chemistry, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn

Michael Famulok

LIMES Program Unit Chemical Biology & Medicinal Chemistry, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn

Version History

CURRENT STATUS: Posted

Version 1

Posted 14 Dec, 2006

MetricsComments: 0HTML Views: ...

Subject Areas

Genetics

Molecular Biology

Biological techniques

DOI:

10.1038/nprot.2006.413

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

10 mg mouse liver (or ca. 10⁶-10⁷ HepG2 cells grown to 75% confluence)

SecinH3 (100 mM in DMSO)

D5 (10 mM in DMSO)

DMSO

insulin (Sigma)

standard cell culture material and media

RNA-extraction kit (Absolutely RNA, Stratagene)

RT-kit (cDNA-Archive kit, Applied Biosystems)

TaqMan probes for assayed genes (Applied Biosystems)

TaqMan MasterMix, 2x (Applied Biosystems)

Standard cell culture equipment

Real-time PCR machine (BioRad)

A: HepG2 cells

ca. 10⁵ HepG2 cells are seeded into wells of a 12-well culture plate and incubated overnight
after washing twice with PBS, the cells are serum starved for 24 hrs
compounds are added in desired concentrations (SecinH3, D5 and Wortmannin or DMSO alone); final concentration of DMSO was 0.2% in our experiment
cells stimulated with 10 nM insulin for 12 hrs
Total RNA extracted with Absolutely RNA kit (Stratagene) according to instructions from manufacturer; elution volume 50 µl

B: Mouse liver

10 mg liver homogenized in lysis buffer and RNA extracted with Absolutely RNA kit (Stratagene) according to instructions of manufacturer; elution volume 50 µl

RT and real-time PCR

RNA concentration determined by photometry
2.5 µg RNA used for reverse transcription with cDNA Archive kit (Applied Biosystems) according to instructions from manufacturer; RT volume: 50 µl
resulting cDNA diluted 1:3 with H₂O
quantitative PCR performed in 10 µl scale with gene-specific TaqMan probes (Applied Biosystems). All expression-levels were normalized to the expression level of β-2-microglobulin.

Associated Publications

Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance
by Hafner, M. et al.
Nature (16 November, 2006)

This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.

Method Article

Genetics

Molecular Biology

+1

Suppression of insulin target gene expression by SecinH3 quantified by real-time PCR

Markus Hafner, Anton Schmitz, Michael Famulok

Markus Hafner

LIMES Program Unit Chemical Biology & Medicinal Chemistry, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn

Anton Schmitz

LIMES Program Unit Chemical Biology & Medicinal Chemistry, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn

Michael Famulok

LIMES Program Unit Chemical Biology & Medicinal Chemistry, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn

DOI:

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Introduction

Reagents

Equipment

Procedure

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Subject Areas

Genetics

Molecular Biology

Biological techniques

Associated Publications

Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance

by Hafner, M. et al.

Nature (16 November, 2006)

Method Article

Genetics

Molecular Biology

+1

Suppression of insulin target gene expression by SecinH3 quantified by real-time PCR

Markus Hafner, Anton Schmitz, Michael Famulok

Markus Hafner

LIMES Program Unit Chemical Biology & Medicinal Chemistry, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn

Anton Schmitz

LIMES Program Unit Chemical Biology & Medicinal Chemistry, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn

Michael Famulok

LIMES Program Unit Chemical Biology & Medicinal Chemistry, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn

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This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

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Associated Publications

Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance

by Hafner, M. et al.

Nature (16 November, 2006)

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